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| Home > Publications database > Untersuchungen zur Biosynthese von Glycin als Vorstufe von Riboflavin in Ashbya gossypii |
| Book/Report | FZJ-2019-03672 |
1998
Forschungszentrum Jülich, Zentralbibliothek, Verlag
Jülich
Please use a persistent id in citations: http://hdl.handle.net/2128/22443
Report No.: Juel-3519
Abstract: The filamentous fungus $\textit{Ashbya gossypii}$ is a vitamin B$_{2}$ overproducer. Enhancement of riboflavin production is known to be achieved by supplementation of the medium with glycine, a precursor of vitamin B$_{2}$ (riboflavin). The present work describes the characterization and deregulation of glycine biosynthetic pathways in $\textit{A. gossypii}$ leading to an improved riboflavin production. In the $\textit{A. gossypii}$ wild type strain ATCC 10895 supplementation with 80 mM glycine lead to an increase in riboflavin production by at least 100 % whereas the growth remained unchanged. Nevertheless only 5 % of the added glycine were consumed during the course of cultivation, which suggested a poor uptake of the amino acid. On unsupplemented media glycine concentration even increased from 1 .9 mM to 3 mM during cultivation. Consequently the effect of glycine on riboflavin production can be attributed to a small net uptake as well as to an inhibition of glycine efflux due to the high extracellular glycine concentration. The glycine biosynthetic enzymes serine hydroxymethyltransferase, threonine aldolase and glutamate glyoxylate aminotransferase were detected in crude extracts of $\textit{A. gossypii}$ with maximum specific activities of 6, 5 and 26 mU/mg protein, respectively. Sucrose density gradient centrifugation of $\textit{A. gossypii}$ organelles showed, that glutamate glyoxylate arninotransferase occurs in the mitochondria of the fungus, thus it is not colocated with isocitrate lyase - the main supplier of glyoxylate - in the peroxisomes. Glycine formation starting from serine and threonine could also be demonstrated $\textit{in vivo}$ using $^{13}$C labelling experiments. Likewise the formation of serine from threonine, which probably proceeds via glycine and therefore means an unwanted loss of glycine, was shown $\textit{in vivo}$. When 70 mnM aminomethylphosphonic acid (AMPS) were added to the culture medium riboflavin production of $\textit{A. gossypii}$ was completely inhibited. Screening on AMPS resistance of riboflavin production lead to the isolation of the strain A.g. AMPS-NM-01. It showed a riboflavin production of 40 mg/g mycelial dry weight (mdw) even in the absence of glycine, which was significantly higher than in the wild type strain (5 mg/g mdw) under the same conditions but resembled wild type riboflavin production in the presence of 80 mM of glycine (30 mg/g mdw). Increased riboflavin production without glycine supplementation suggested a better intracellular availability of glycine in the mutant strain. Nevertheless, even in this case riboflavin production could be increased by glycine supplementation to 95 mg/g mdw. In comparison to the wild type strain serine hydroxymethyltransferase specific activity was significantly reduced from 3 to 1.5 mUlmg protein in the strain A.g. AMPS-NM-01. Therefore the increased riboflavin production of this strain can be explained by a better intracellular availability of glycine conditioned by a reduced loss of glycine for the formation of serine. Using heterologous complementation of a $\textit{Saccharomyces cerevisiae}$ mutant auxotrophic for glycine a GLY1 homologous gene with unknown function was isolated from $\textit{A. gossypii}$. Characterization of the corresponding enzymatic activity showed that the isolated gene as well as the GLY1 gene from $\textit{S. cerevisiae}$ encode a threonine aldolase. In contrast to $\textit{S. cerevisiae}$ the GLY1 knock-out mutant of $\textit{A. gossypii}$ was not auxotrophic for glycine, which demonstrated that threonine aldolase plays only a minor role during glycine biosynthesis of $\textit{A. gossypii}$. GLY1 was overexpressed in $\textit{A. gossypii}$ under the control of the TEF-promotor and -terminator using the expression vector pAG203. In crude extracts of A.g. pAG203GLY1 50 mU/mg protein of threonine aldolase specific activity were detected indicating a tenfold overexpression in comparison to the wild type. When 50 mM threonine was fed to A.g. pAG203GLY1 an increase in riboflavin production from 2 to 16 mg/g mdw was determined, an increase never reached with glycine because of its worse uptake. Threonine was found to be taken up efficiently by this strain. Its conversion to glycine was confirmed by a striking efflux of glycine into the medium. Extracellular glycine correspondingly increased from 2 to 44 mM. The requirement to feed threonine in addition to threonine aldolase overexpression demonstrated a limitation in threonine biosynthesis, which was confirmed by feeding experiments with threonine precursors.
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